Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响*

doi:10.3969/j.issn.1672-5069.2014.05.019

实用肝脏病杂志 ›› 2014, Vol. 17 ›› Issue (5): 523-527.doi: 10.3969/j.issn.1672-5069.2014.05.019

Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响^*

王晖晖, 张峰, 沈珊珊, 诸葛宇征

210009南京市东南大学医学院（王晖晖）; 南京大学医学院附属鼓楼医院消化科(张峰,沈珊珊,诸葛宇征)

收稿日期:2014-02-07 出版日期:2014-10-31 发布日期:2016-04-11
通讯作者: 诸葛宇征,E-mail：yuzheng9111963@aliyun.com
作者简介:王晖晖,女,24岁,硕士研究生。E-mail:wanghuihui19891021@163.com
基金资助:
江苏省自然科学基金资助项目（BK2011094）

Effects of Rac1 on epithelial-mesenchymal transition,and proliferation and apoptosis of L02 cells in vitro

Wang Huihui, Zhang Feng, Shen Shanshan

School of Medicine,Southeast University,Nanjing,210009,Jiangsu Province,China

Received:2014-02-07 Online:2014-10-31 Published:2016-04-11

摘要/Abstract

摘要： 目的检测Rac1在TGF-β1诱导的L02细胞上皮间质转化中的作用,及其对细胞增殖和凋亡的影响。方法应用不同活性的Rac1质粒pExRed-NLS Flag（空载体组）、pExRed-NLS Flag Rac1（野生型Rac1组）、pExRed-NLS Flag Rac1T17N（显性负调控Rac1组）、pExRed-NLS Flag Rac1G12V（持续活化型Rac1组）瞬时转染L02细胞,经5 ng/ml TGF-β1处理细胞。采用免疫印迹法检测融合蛋白Flag-Rac1表达,采用细胞免疫荧光及免疫印迹法检测Ck8和Vimentin表达,采用细胞划痕实验及Transwell法检测细胞迁移能力。使用不同浓度的Rac1特异性抑制剂NSC23766处理L02细胞,采用CCK-8法检测细胞增殖,采用Annexin V-FITC/PI双染法检测细胞凋亡。结果四组质粒均成功瞬时转染到L02细胞中;与空载体组和野生型Rac1组比,持续活化型Rac1转染细胞Vimentin蛋白表达水平显著增高,CK8蛋白表达水平降低,细胞迁移能力增加;与空载体组和野生型Rac1组比,显性负调控Rac1转染细胞Vimentin蛋白表达水平降低,CK8蛋白表达水平增高,细胞迁移能力降低（P<0.05）;在NSC23766处理L02细胞后,细胞增殖被抑制,但各处理组细胞凋亡无明显差异。结论Rac1可促进TGF-β1诱导的L02肝细胞株上皮间质转化和细胞增殖,但对细胞凋亡无明显影响。

关键词: L02细胞, Rac1, 上皮间质转化, 增殖, 凋亡

Abstract: Objective To investigate the effects of Rac1 on epithelial mesenchymal transition（EMT） induced by transforming growth factor β1（TGF-β1）,and on cell proliferation and apoptosis of L02 cells in vitro. Methods Rac1 plasmids with different activity including pExRed-NLS Flag（vector）,pExRed-NLS Flag Rac1（wild type）,pExRed-NLS Flag Rac1T17N（dominant negative mutant） and pExRed-NLS Flag Rac1G12V（constitutively active mutant） were transiently transfected into L02 cells,followed by stimulation of exogenous TGF-β1 at dose of 5ng/ml;Exogenous Flag-Rac1 fusion protein was determined by Western blot analysis;Immunofluorescence and Western blot were used to evaluate epithelial markers of Ck8 and mesenchymal markers of vimentin;Cell motility was assessed by transwell assay and wound healing assay;L02 cells were treated with different concentrations of Rac1 inhibitor （NSC23766）,and the influence of Rac1 on cell proliferation and apoptosis were detected by CCK-8 assay or Annexin V-FITC/PI double staining,respectively. Result All kinds of plasmids were successfully transiently transfected into L02 cells;Compared with the vector group and the wild type group,constitutively active mutant pExRed-NLS Flag Rac1G12V significantly increased the expression of vimentin,decreased the expression of CK8 and enhanced cell motility,whereas the effects of dominant negative mutant pExRed-NLS Flag Rac1T17N were just the opposite of above results（P<0.05）;Disruption of Rac1 activity with NSC23766 inhibited cell proliferation（P<0.05） without increasing cell apoptosis. Conclusions Rac1 promotes the EMT process and cell proliferation induced by TGF-β1 in L02 cells without affecting cell apoptosis in vitro.

Key words: L02 cells, Rac1, Epithelial-mesenchymal transition, Proliferation, Apoptosis

王晖晖, 张峰, 沈珊珊, 诸葛宇征. Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响^*[J]. 实用肝脏病杂志, 2014, 17(5): 523-527.

Wang Huihui, Zhang Feng, Shen Shanshan. Effects of Rac1 on epithelial-mesenchymal transition,and proliferation and apoptosis of L02 cells in vitro[J]. JOURNAL OF PRACTICAL HEPATOLOGY, 2014, 17(5): 523-527.

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Rac1对L02肝细胞株上皮细胞间质转化及增殖和凋亡的影响^*

Effects of Rac1 on epithelial-mesenchymal transition,and proliferation and apoptosis of L02 cells in vitro

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